Project Summary/Abstract Many human diseases arise from mutations that disrupt the cell?s normal behavior, such as in cancer and neurodegenerative disorders. One method to study the function of genes is to ablate their function using genome editing and study the outcome in animal models. Clustered regularly interspaced palindromic repeats (CRISPR) associated proteins, such as Cas9, have emerged as the preferred genome editing tool for both healthcare and life science applications because it is simple to use compared to other methods. Cas9 is an endonuclease derived from prokaryotes that is guided to a 20-nucleotide sequence in the genome called the protospacer by a guide RNA. Cas9 can be programmed by changing the sequence of the guide RNA making it a programmable DNA cutting protein. This technology is already widely used in the life sciences to study gene function, and the first Cas9 based drugs are entering phase-1 clinical trials. However, inefficient activity in mammalian cells is a bottleneck preventing widespread usage. Cas9 specificity enhanced variants and activity optimized and guide RNA have been developed to improve activity. We and other groups have studied how Cas9 variants and different guide RNAs influence DNA binding, unwinding and cleavage. Despite the importance of proper Cas9 complex assembly, Cas9 complex binding and Cas9 cleavage of DNA in a chromatin compacted mammalian cell, our understanding of the molecular details of these processes and is poor. In two specific aims, I propose to fill in these knowledge gaps by first quantifying and observing in real- time how Cas9 complex assembles, and its activity of nucleosomes. Aim one is to adapt a previously developed single molecule assay where I can mimic co-transcriptional RNA folding to studying the molecular steps of Cas9 assembly with the guide RNA as it folds. Aim two will probe how Cas9 can binds and cleaves DNA as a function of DNA flexibility around nucleosomes using a high-throughput sequencing assay. I will then quantify the binding kinetics and equilibrium constants as a function of DNA flexibility around nucleosomes. The results of these experiments will significantly contribute to our fundamental understanding of CRISPR Cas enzymes and will aid efforts to develop Cas9 based therapeutics for cancers and neurodegenerative diseases. Accomplishing these aims will also provide technical training in multicolor single molecule FRET, biochemistry, molecular biology and next-generation sequencing. Furthermore, analyzing data, writing manuscripts summarizing my findings, and presenting at conferences will enhance quantification and soft skills. The Ha laboratory and Johns Hopkins University are excellent environments for this research training mainly because of the access to a broad range of expertise and to resources.